05). Quantification of leaf-associated survival Leaf-associated fitness was evaluated as previously described [51]. Briefly, overnight cultures in SB medium were centrifuged to recover bacteria cell pellets, washed and resuspended in 10 mM phosphate buffer (pH 7.0) at a concentration of 109 CFU/ml. These bacterial suspensions were sprayed onto leaves until each leaf surfaces were uniformly covered. Old citrus leaves were used since the greater thickness of the cuticles of these leaves naturally AZD1480 datasheet render

the leaves resistant to bacterial entry (unpublished results). Four different leaves were inoculated with each strain, leaves were photographed and the surfaces were quantified using the software Image-Pro (Media Cybernetics). Leaves were collected on different days post-inoculation and transferred to borosilicate glass flasks containing 10 mM potassium phosphate buffer (pH 7.0). Flasks were submerged learn more in a sonicator (Branson model #5510) for 10 min. Subsequently, each flask was vortexed for 5 sec, bacteria were recovered by centrifugation and serial dilutions were plated on SB plates containing Ap to count bacterial colonies. Results were expressed in CFU/cm2 of inoculated leaves. Values represent an average

of four leaves assayed for each strain, the data were statistically analyzed using one-way ANOVA (p < 0.05). RNA preparation and RT-qPCR Total RNA from bacterial cultures grown at the indicated conditions and from bacteria recovered from leaves at the indicated

times were isolated using TRIzol® reagent (Invitrogen), according to the manufacturer’s instructions. The RT-qPCRs were performed as previously described [52] with the specific oligonucleotides detailed in Additional file 3: Meloxicam Table S1. As a reference gene, a fragment of 16S rRNA (XAC3896) was amplified using the same RT-qPCR conditions. To control that no bacterial DNA contamination was present in the samples, the same PCR reactions were carried out without retrotranscription and non amplification was observed. To ascertain the absence of plant RNA in bacterial samples controls with plant actin primers were carried out (data not shown). Values were normalized by the internal reference (Ctr) according to the equation ΔCt = Ct – Ctr, and quantified as 2–ΔCt. A second normalization using a control (time = 0 days) (Ctc), ΔΔCt = Ct – Ctc, producing a relative quantification: 2–ΔΔCt[53]. Values are the means of four biological replicates with three technical replicates each. Results were analyzed by Student t-test (p < 0.05) and one-way ANOVA (p < 0.05). Protein extraction and resolubilization for the proteomic analysis Biofilms of statically grown bacterial cultures were obtained as previously described [42]. After seven days of static growth, the XVM2 medium was carefully removed and biofilms were collected by pipetting, transferred to a new tube and pelleted by centrifugation prior to protein extraction.